Light- induced electron transfer and ATP

synthesis in a carotene synthesizing

insect
Jean Christophe Valmalette
1
, Aviv Dombrovsky
2,4
, Pierre Brat
3
, Christian Mertz
3
, Maria Capovilla
4
& Alain Robichon
4
1
IM2NP UMR 7334 CNRS, Universite du Sud Toulon Var, P.O. Box 20132, 83957 La Garde CEDEX, France,
2
Volcani Center,
Institute of Plant Protection, P.O. Box 6, 50250 Bet Dagan, Israel,
3
CIRAD UMR QualiSud, 73 rue J.F. Breton, TA B-95/16, 34398
Montpellier CEDEX 5, France,
4
UMR7254INRA/CNRS/UNS, Institut SophiaAgrobiotech, 400route des Chappes, P. O. Box 167,
06903 Sophia Antipolis, France.
A singular adaptive phenotype of a parthenogenetic insect species (Acyrthosiphon pisum) was selected in
cold conditions and is characterized by a remarkable apparition of a greenish colour. The aphid pigments
involve carotenoid genes well defined in chloroplasts and cyanobacteria and amazingly present in the aphid
genome, likely by lateral transfer during evolution. The abundant carotenoid synthesis in aphids suggests
strongly that a major and unknown physiological role is related to these compounds beyond their canonical
anti-oxidant properties. We report here that the capture of light energy in living aphids results in the photo
induced electron transfer from excited chromophores to acceptor molecules. The redox potentials of
molecules involved in this process would be compatible with the reduction of the NAD
1
coenzyme. This
appears as an archaic photosynthetic systemconsisting of photo-emitted electrons that are in fine funnelled
into the mitochondrial reducing power in order to synthesize ATP molecules.
C
arotenoids are initially formed by the condensation of two geranylgeranyl diphosphate molecules (GGPP,
C20) joined at their ends
1,2
. The resulting lycopene, a key intermediate linear molecule, is then modified by
the addition of a carbon ring at one or both ends (see figure 1). Carotenoids constitute a large group of
compounds (the chemical structure of 750 derived molecules has been determined from plants, algae, bacteria
and invertebrates and diverges by minor modifications)
3
. The addition of epoxy groups or alkyl groups to the
basic structure generates the predominant derivatives. These modifications added to the multiple cis/trans
isomers of the polyene double bonds in the aliphatic chain explain the vast repertoire of the carotenoid family
4,5
.
Moreover, a large number of derivative products, like the conjugated complex epoxy carotenoid/a-Tocopherol
and multiple esterified forms with hydrophilic glycosilic groups, have been reported
4,6
. The carotene basic
structure is highly hydrophobic and included in the lipid bilayer. On the other hand, non-covalent binding to
haemolymph circulating proteins, which makes themwater soluble and/or transportable, represents a substantial
fraction of the total carotenoid load in aphids
7,8
. These molecules display yellow to orange colours but chemical
modification and/or protein binding can trigger changes towards green or brownish
7
. Their functions are well
documented in plant photosynthesis where they harvest light energy for the chlorophyll and are scavenger for
reactive singlet oxygen
5
. One other striking function of carotene derivatives is that their cleavage leads to retinal
which is part of eye photoreceptors in all the taxa
6
.
Acyrthosiphon pisum aphids reproduce parthenogenetically in spring and summer whereas sexual morphs
emerge only in autumn
9,10
. We have shown that epigenetic mechanisms in clonality context can be recruited in
order to achieve fitness in unfavourable climate conditions, concomitant in some cases with colour changes
11
.
More recently, authors have reported that the aphid genome harbours the genes required for carotenoid synthesis
like in plants, algae and fungi, which makes this insect species unique in the insect class
12
(see Supplementary
Data, figures S1 and S2). Aphids seemequipped for processing the full carotene synthesis instead of taking it from
their diet (aphids suck the plant phloem, which a priori excludes the uptake of the hydrophobic carotenoid
molecules). A. pisum therefore can exhibit an heavy load of carotene conferring strong orange colour depending
on the environmental context. However, many other pigments are synthesized in this species as well. The best
known molecules are the polycyclic/polyphenolic aphins derivatives (xantho-, erythro- and proto-aphins), which
display red (alkaline pH) or yellow (neutral pH) colours and strong yellow fluorescence when they are excited by
SUBJECT AREAS:
ENVIRONMENTAL
CHEMISTRY
GENE FLOW
METABOLISM
PLASTICITY
Received
14 March 2012
Accepted
19 July 2012
Published
16 August 2012
Correspondence and
requests for materials
should be addressed to
A.R. (robichon@
sophia.inra.fr)
SCIENTIFIC REPORTS | 2 : 579 | DOI: 10.1038/srep00579 1
UV light
7
. Their physiological roles in aphids and other insects are
yet poorly documented. In this report, we analyzed the carotenoid
components in few variants presenting singular pigmentation
(greenish, orange and pale yellow/white). The aphid descendants
of a unique founder mother were placed in different unfavourable
environments in order to select viable and robust variants in clonality
context
11
. Surprisingly, a cold adapted strain was obtained after a
process of selection and presents a singular greenish pigmentation.
The panel of carotene molecules found in the green and orange
phenotypes has been extensively analyzed by mass spectrometry
after extraction/chromatographic isolation and also by Raman
imaging technology directly on living aphids to reveal the carotene
signature. Putative physiological functions that might use carotenoid
molecules have been investigated. The free electrons generated by
photo-activated carotenoids and/or other pigments appeared to be
transferrable to the reducing power machinery [like the reduction of
NAD(P)1 to NAD(P)H)] followed by a subsequent mitochondrial
ATPsynthesis. This report describes experimental data that argue for
a role of chromophores as part of an archaic photosynthetic mech-
anism in insects.
Results
Ten orange adult aphids were placed each day at 8uC for five months
before we obtained a viable and robust colony of green variants
(figure 1). By chance, progenies of the orange adult aphids did not
survive at 8uC. Most larvae died from larval stage 1 to stage 4. Cold
(8uC) conditions successfully selected a viable and robust green aphid
lineage from orange descendants of a 22uC-adapted unique founder
mother. Importantly, the switch to the cold adapted green variant
never occurred within the actual orange adults, which excludes a
direct enzymatic induction. The green phenotype is heritable in the
conditions in which it emerged, but its singular pigmentation fades
away when it is placed back in optimal conditions at 22uC. This
demonstrates that a clonal aphid population under pressure of
selection is able to generate complex traits guiding environmental
fitness and underlying the recruitment of gene networks
11
. The scen-
ario precludes allele selection as an explanation (success of the
phenotypic adaptation too fast for implying a Darwinian process)
and strongly supports the hypothesis of an epigenetic regulation. The
mechanism might reside in the extensive DNA methylation as the
molecular cue to transmit complex traits in the framework of an
unchanged genome
11
. This selection process is summarized in fig-
ure 1. On the other hand, if the pink/orange pigmentation is dom-
inant at 22uC in optimal conditions (low population density and
abundant resources), the declining conditions (rarefaction of
resources, high population density) trigger the progressive dis-
appearance of the pink/orange phenotype and its replacement by
pale/white/yellow colours (figure 1). In such case, the colour plas-
ticity (colour shift orange to pale/white/yellow) is proportionally
induced by the increase of population density and the rarefaction
of resources. The pale/white/yellow phenotype reflects an unfavour-
able environment and might be referred as survival forms that have
turned down some less essential biochemical processes to minimize
energy cost.
Following the intriguing discovery of the carotene synthesis genes
in the aphid genome, we undertook an extensive analysis of carot-
enoid molecules by Raman spectrometry imaging and mass spec-
trum technology in the framework of this genetic/epigenetic context.
We took advantage of the rapid crystallisation of carotene molecules
to isolate and to solubilise them in ethanol/acetone. A long centrifu-
gation (9,300 x g for 1 hour) of PBS buffered extract of aphids trig-
gers the formation of a pure orange crystallized precipitate at the top
of the aqueous phase. Spectral absorbance properties of this precip-
itate were analyzed and were found to be in accordance with carot-
enoid molecules. A comparative absorbance profile between the
extracts from the green and pale orange phenotype is presented in
figure 2. As expected, the decrease of spectral absorbance of the pale
aphid acetone/ethanol extract in the wave lengths of carotenoid
Figure 1 | Protocol scheme used to generate the green variant. The graph on the left summarizes the protocol used to select the green variant from a
unique parthenogenetic founder mother. The photographs show the differences between the green, orange and pale adult aphid phenotypes. The white
phenotype is directly induced by high population density and rarefaction of resources. The green phenotype was the result of a process of selection in cold
conditions. All these variants were the descendants of a unique orange founder mother adapted to optimal conditions (22uC). Lycopene, b-carotene and
c-carotene are represented.
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SCIENTIFIC REPORTS | 2 : 579 | DOI: 10.1038/srep00579 2
molecules absorption was spectacular compared to the green aphid
extract (Figure 2).
Imaging of resonance Raman spectrometry allowing non-
destructive molecular motif identification and quantification was
performed to detect structural elements of carotene molecules
directly on living aphids. A 488 nm laser wavelength corresponding
to the maximum wavelength of carotene absorption was used to
excite the polyene motifs (Raman conditions: 1.2 to 12 mW/13
seconds). The shift values [the shifts of the Stokes (lower energy)
and Anti-Stokes (higher energy) Raman light scattering correspond
to a vibrational mode of a structural motif in a molecule] are
expressed as cm
21
. The carotene signature corresponding to the three
peaks obtained at 1,520 cm
21
(assigned to the C5Cstretching vibra-
tion), 1,157 cm
21
(CH-CH) and 1,005 cm
21
(CH-CH3) were always
found in living aphids even though these molecules are part of a
complex biological matrix (figure 3). The laser beam of the Raman
imaging apparatus was also directed on the crystals spontaneously
formed after crushing adult orange aphids (figure 3). Moreover a
Raman imaging control was carried out using the reddish/brown
aphid eyes, known to contain, as any eye in all the taxa, a strong
concentration of retinal. Retinal (vitamin A) conserves the structural
motifs of carotene by the fact that it is the enzymatic conversion
product of carotenoid molecules. The three peaks corresponding to
the Raman signature of carotene were unambiguously obtained with
living aphids and crystals, which suggests high concentration of these
compounds (figures 3 and 4). Interestingly, a stronger intensity of the
peaks was consistently found with the green compared to the orange
phenotype (40% increase) (figure 4). Moreover, the method was able
to followthe carotene synthesis in the developing embryos where the
signals were correlated with the apparition of the orange pigmenta-
tion (see Supplementary Data, figure S3).
The extensive comparative analysis of these molecules between the
green and the orange aphids has been performed by mass spectro-
metry (after chromatographic isolation), in order to quantify few
intermediate components in the cascade of carotenoid synthesis.
The major components of the carotene family found in the green
and orange aphids is reported in table 1. A substantial increase of
concentration of trans-b and trans-c carotene is observed in the
green variant compared to the orange (8.36 1.2 versus 4.0 6 1.9
and 12.661.5 versus 6.561.8 mg.100 g
21
respectively). At the oppos-
ite, the cis-torulene is drastically increased in the orange phenotype
(10.7 6 3.4 versus 3.0 6 0.6 mg.100 g
21
) whereas the trans-torulene
(a precursor metabolite) was roughly unchanged (Table 2).
Therefore, the mass spectrum analysis confirmed the trend observed
by Raman analysis (see Supplementary Data, figure S4).
One well documented role regarding these compounds are the
annihilation of singlet oxygen and radical scavengers in plant pho-
tosynthesis along with the light harvesting function of chlorophyll
5
.
Figure 2 | Absorbance properties of green, orange and pale aphid ethanol extracts. The orange pigments were extracted as indicated in Methods. Briefly,
250 mg of adult aphids were centrifuged in Ringers buffer and the orange layer was collected. This precipitate was then solubilized in ethanol/acetone
(75/25). Acomparative spectrumof absorbance was carried out from320 to 700 nm(A), at 600 nm, 650 nmand 700 nm(B) and at 425 nm, 450 nmand
480 nm (specific peaks of carotene absorbance) (C). A comparative measure was carried out at 295 nm (peak of absorbance of phytoene, a precursor of
carotene) and at 450 nm(peak of carotene absorbance) with the orange crystallized precipitate obtained with the green and orange aphid extracts (D). The
means of three independent experiments are shown in (A) and bars in (B), (C), (D) represent the average of three experiments 1/2 S.E (comparison
orange versus green, P, 0.002). More pigment is present in green than in orange aphids.
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SCIENTIFIC REPORTS | 2 : 579 | DOI: 10.1038/srep00579 3
Because strong carotenoid concentration was observed especially in
the green and orange larval forms (despite a high level of variation
between individuals in the same colonies when food resources are
declining), we tried to unravel some putative physiological functions
beside their canonical anti-oxidant properties. We investigated the
hypothesis that the photon energy might excite the p delocalized
electrons of the carotene polyene structure and trigger an electron
transfer to acceptor molecules. Adult aphids (green, orange and white
phenotypes) and larvae from orange mothers were placed in dark
or left in light photoperiodicity (18/6 hours). Furthermore, these
conditioned aphids were returned under light photoperiodicity
(18/6 hours) after a dark episode. The striking data show that ATP
synthesis is sensitive to light, but differs among the orange (marked
effects), green (little effects likely because a strong lipid load in this
variant acts as a metabolic reserve) and white phenotypes (no
change). Results are summarized in figure 5 and support the concept
of photo-conditioning of ATP synthesis in some environmentally
shaped variants.
To get more insight about the light-dependent reduction-oxida-
tion (redox) process, orange aphid extracts were used to reduce tetra-
zolium salts (MTT) in presence or absence of light. Although the
effect was moderate, an increase of MTT reduction in presence of
light was obtained with the orange, but not with the white extract
(figure 6). This trend was also obtained with orange embryos incu-
bated with MTT and exposed to light whereas the white embryos in
the same conditions display a weak fluctuation of the basal level
(figure 6). The same results were observed when the experiments
were conducted with pure molecules. Briefly, 100 ml of MTT solubi-
lized in water were placed on a layer of dry b-carotene and illumi-
nated by a regular electric light. The reduction of MTT in blue
precipitated formazan was observed as the result of a capture of free
electrons generated by the photoactivated carotene, which suggests
that the energy of these free electrons is high enough to pass the
barrier of the tetrazolium redox potential (see Supplementary
Data, figure S5).
Finally the balance NAD
1
/NADH was measured in the light ver-
sus dark context. A series of experiments shows unambiguously a
Figure 4 | Carotene signature in aphid adults corresponding to the green
and orange phenotype by Raman imaging. Individual orange (A) and
green (B) living aphid phenotypes (see figure 1 for details) were analysed
according to the same protocol as in figure 3. Each assay was conducted
with individual adult aphids ten days after the birth of the first instar larva.
A significant increase of the intensity of the three identified signals (1,550,
1,150 and 1,005 cm
21
shifts) was consistently observed for the green
phenotype indicating a stronger carotenoid pigmentation. Each colour
represents the measures with different individual adult aphids. Raman
conditions: 488 nm; 1.2 mW; 1 s.
Figure 3 | Carotene signature in aphid eyes and spontaneous crystals by
Raman imaging. A 488 nm laser excitation of the Raman spectrometry
was used. A control with pure b-carotene is shown. Two other spectra are
shown: the microscope laser was focused on an eye and on spontaneous
crystals obtained after crushing adult orange aphids in PBS solution. The
1,550, 1,150 and 1,005 cm
21
shifts correspond respectively to the C5C,
CH-CH and CH-CH3 motifs. Conditions of Raman: 488 nm; 1.2 mW;
3 s. The panels at the top represent spontaneous crystals, an eye, and
embryos (mature orange embryos in a germaria plus an ovariole stained in
green with anti HRP
10
). b-carotene is present in whole aphids and in aphid
eyes.
Table 1 | Comparative quantification of carotenoid molecules in the
green and orange aphid phenotype by LC-MS/MS. Chara-
cteristics of identified carotenoids of orange and green aphids:
the molecules were extracted as indicated in Methods and then
submitted to HPLC separation and mass spectrumanalysis. Strong
differences are observed between the two environmentally
selected variants originated from a unique parthenogenetic
founder mother. No significative differences were observed for
all-trans-torulene and 3,4-dehydrolycopene as they were for the
other compounds analysed
Peak Tr (min) l
max
% III/II M Compound*
1 35.0 (425), 453, 477 15 536 all-trans-b-carotene
2 46.3 436, 463, 492 40 536 all-trans-c-carotene
3 52.7 380, 458, 483, 515 43 534 cis-torulene
4 59.3 378, 463, 488, 520 50 534 all-trans-torulene
5 63.5 380, 465, 492, 525 60 534 3,4-dehydrolycopene
*
Tentative identification.
Table 2 | Quantification (mg.100 g
21
) of carotenoids of orange
and green aphids
Compound Green aphids Orange aphids
all-trans-b-carotene 8.3 6 1.2
a
4.0 6 1.9
b
all-trans-c-carotene 12.6 6 1.5
a
6.5 6 1.8
b
cis-torulene 3.0 6 0.6
a
10.7 6 3.4
b
all-trans-torulene 9.1 6 1.3
a
9.9 6 1.5
a
3,4-dehydrolycopene 5.7 6 1.7
a
9.1 6 3.9
a
Values are means 6 SD of three independent determinations. Values within a row labeled with
different letters are significantly different (NewmanKeuls, P , 0.05).
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SCIENTIFIC REPORTS | 2 : 579 | DOI: 10.1038/srep00579 4
significant increase of the reduced co-enzyme level in particulate
fraction enriched in mitochondria when the orange aphids are
exposed to light (figure 7). Intriguingly, a drastic decrease in
NAD
1
(oxidized) concentration was found in the soluble fraction
of the extract when the orange aphids were maintained in dark,
suggesting that its synthesis is partly controlled by light. By contrast
and as expected, the white aphid variants display weak levels of
NAD
1
/NADH, which seem little affected by light (figure 7).
Together these data reinforce the hypothesis that light, through bio-
logical membranes enriched in pigments, triggers a reducing power
that in fine is captured by co-enzymes like NAD
1
. This reduced co-
enzyme is known to transit inside mitochondria through a shuttle
mechanismand deliver electrons for the respiratory chain machinery
ending with the H
1
inflow-driven ATP synthesis
1315
. Amazingly, we
observe that carotene molecules are disposed as a bilayer under the
cuticule from 0 to 40 mM in depth, suggesting that this structure
might present an optimal efficiency to harvest light energy (figure 7).
Discussion
Aphid lycopene cyclase and phytoene synthase are enzymes fused in
one unique protein with two distinct catalytic activities. This rare
hybrid has been previously described in Phycomyces
16
. Surprisingly,
to our knowledge genes like phytoene dehydrogenase and lycopene
cyclase/phytoene synthase, key enzymes in the cascade of carotenoid
synthesis, seem to be present in different locations of the aphid
genome (see Supplementary Data, figure S1).
The existence of this fused gene in the aphid genome highlights the
regulation of the two enzymatic activities by a unique promoter. This
suggests a probable in situ carotene synthesis and at this stage makes
the aphids unique in the insect class
12
. On the other side, the avail-
ability of free carotenoid pigments in phloem sap on which the
aphids feed is very unlikely, because of their hydrophobicity. Our
data suggest strongly that the environmentally-guided synthesis of
these compounds in aphids plays a role in absorption of sun light and
in electron transfer to mitochondrial protein complexes. This is cor-
roborated by the fact that the emergence of sexuals, life history traits
and metabolism are highly dependent on photoperiodicity in this
species
9,10
. To argue in favour of this scenario, the photoconditioning
of the Pieris brassicae caterpillar mediated by pterobilin, an abundant
pigment found in their integument, has been described to drive a
light-dependent production of ATP
17
. Moreover, the accumulation
of carotenoid compounds in different caterpillar species has been
well documented and currently associated with canonical anti-
oxidant properties, although other putative functions have not been
investigated up to date
18,19
. Insects, except aphids, do not synthesize
these molecules, but absorb them by food uptake from the chloro-
plasts of plants/algae
1,2
. The polyenic-conjugated structure of caro-
tenoids (C5Calternated with C-Cleading to p delocalised electrons)
Figure 5 | ATP dosage in dark conditioned aphids. (A). Dark exposure of adult aphids. Aphids were placed in dark, then tested after two days (1 and 2; 5
and 6) or alternatively were kept in light two days more as control (3 and 4; 7 and 8) before the measure of ATP content. 14 and 58 are the separate ATP
determinations obtained with green aphids and orange aphids. a and b: ATP dosage determined with the content of 5 and 1 aphids, respectively. c: ATP
determination obtained with the content of one white aphid. The standards roughly represent 50 pmoles (blue) to 250 pmoles (red). (B). Dark exposure of
larvae. Larvae from orange aphid were kept two days in light (a) or alternatively two days in dark (b). A control white larva aphid kept two days in light
after birth is shown(c). The determinations were done with 5 larvae. (C). Comparative time course of the decline of ATPcontent in embryos and larvae placed
in dark. (see methods for experimental design). (D) Light-induced ATP synthesis after a dark exposure episode of orange adult aphids. 1, 2 are ATP dosages
from separate experiments. (a) light-exposure control. (b) and (c) are the dark exposure for two and three days, respectively. 3 and 4 are orange adult
aphids placed two days in dark (a, b, c). 5 and 6 are the same aphids than in 3 and 4 placed back in light for one day (a) or kept one day (b) or two days more
(c) in dark. 7 and 8 are the light rescue of ATP content three days after a dark exposure of two days (c) versus the light control (a). (E) Comparative
determination of ATP between green, orange and white adult aphids in dark or light. Bars represent the mean of three different experiments 1/2S.E.
(** P,0.005). (F) Light-induced ATP synthesis after a dark exposure episode in larvae. Top: First instar larvae were tested at day 0 (1), at day 2 (2) and day 3
(3). Down: Emerged progenies were tested at day 0 (1), at day 2 (2) and finally at day 3 (3).
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SCIENTIFIC REPORTS | 2 : 579 | DOI: 10.1038/srep00579 5
appears an efficient mode to transport electrons across the lipid
bilayer, putatively funnelling them to the mitochondrial redox
machinery. This might end up with another level of regulation of
ATP synthesis. The electron transport chain might partly use the
electrons from photo-activatable pigments. The linear polyene motif
of carotene might act as a molecular wire in electron transfer to
acceptors across the bilayer membranes. Furthermore, aphids syn-
thesize other pigments like the aphins for which the physiological
roles are little known
9
. Their redox properties based on a polyphe-
nolic structure and the inherent p delocalized electrons make them
solid candidates to fulfill the same functions as those attributed to
carotenoids (see Supplementary Data, figure S6). A large panel of
pigments might act as a light and energy harvesting systemleading to
a photon-triggered electron separation and subsequent transfer to an
acceptor. To corroborate this hypothesis and to confirm our data,
the photoreduction of NAD
1
by light-excited chromatophores,
extracted from a purple colored proteobacterium that synthesizes
carotenoids, has been reported
20
. Furthermore and independently,
the photoreduction of NAD
1
in presence of metal complexes and
organic compounds that donate electrons has been also described
21
.
An artificial photosynthetic system like self-assembling of a mix of
phenyl dipeptides, porphyrin and metal as platinium was success-
fully tested to transfer electrons to NADP
1
via light excitation
22,23
.
Finally, the photopotential and photocurrent generated by carotene
molecules and chlorophyll have been compared and authors report
that the efficiency of electron transfer to an electrode was higher with
the illumination of carotene than of chlorophyll
24
. In parallel experi-
ments, by building triad molecules (carotene, porphyrin, quinone
and/or fullerene), authors have generated a strong electron separa-
tion from carotene (after light absorption) for the benefit of
the quinone or fullerene component
25
. This chemical system was
designed as a model to study the redox properties of carotene because
the photo-excited electrons in this compound are extremely short
lived to be tracked
25
. To this regard, a systemcomposed of b-carotene
placed inside carbonnanotubes was used to study the light harvesting
and chemical capture of energy
25
. Moreover and more importantly,
the photoconversion of GFP protein (concomitant to a shift from
green to red color) and the photo-induced electron transfer from
GFP to tetrazolium, quinone, FMN
1
and NAD
1
has been observed
and reported
26
. The redox potential of NAD
1
is quite high
[Eu520,32 v], which suggests that many proteins such as GFP
under light absorption are able to donate electrons with a level of
energy compatible to pass the redox barrier of NAD
1
. Therefore, the
auto fluorescence of insects coming from numerous endogenous
molecules might be concomitant with electron separation, captured
subsequently by oxidized co-enzymes. The aphins (erythoaphin,
xanthoaphin and proto aphins), which are polycyclic and polyphe-
nolic compounds known to complex metal as Fe
21
, might participate
Figure 6 | Tetrazolium (MTT) reduction by orange aphid extract. 100 ml of tetrazolium solution (1 mM in water) were placed on a glass slide in which
10 ml of orange aphid extracts were added. The system was irradiated by visible light (A) for 30 min or kept in dark (B). Then, the medium was delicately
washed out. Top: The photos show the border of the spots where the formazan precipitation is more intense. Unambiguously, an increase of MTT
reduction, measured as formazan precipitation on the glass, is observed under light (A). Middle: higher magnification of the photograph above. Bottom:
The light exposure of strongly pigmented ovarioles in presence of MTT (1 mM in water) is compared with white/pale ovarioles in the same conditions as
above. Produced formazan by orange or white aphid extract (100 mg protein) under light or kept in dark was measured after solubilization in acid/ethanol
(C and D). The representations are the mean of three separate experiments.
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SCIENTIFIC REPORTS | 2 : 579 | DOI: 10.1038/srep00579 6
to this phenomenon
7
. We suggest that these complex pigments con-
stitute a system of photo-induced electron transfer that (due to their
high energy) are able to reduce efficiently NAD
1
bound to mem-
brane proteins ending in fine with ATP synthesis.
Methods
Maintenance and propagation of aphids. The pea aphid Acyrthosiphon pisum
belongs to the order Homoptera (Aphididae family) and feeds on the Vicia faba plant.
Aphids were maintained on V. faba in cages in an incubation room at about
22uC1/23uCand a light/dark photoperiodicity of 16/8 hours. Aphids were raised at
8uC to select a predominant phenotype (green body color). The white phenotype was
induced immediately by increased density of population and by declining plant
resources at room temperature.
Reagents and commercial kits for ATP dosage and NAD
1
/NADH assay. Thiazolyl
blue tetrazolium bromide (MTT) was purchased from Sigma Aldrich. The balance
and determinations of NAD
1
/NADH were carried out with the ultrasensitive
colorimetric kit purchased from Bioassay Systems, Hayward CA USA (catalog n.
ECND-100). Finally the ATP dosage was performed with the kit FLASC purchased
from Sigma Aldrich.
ATP determination: Detailed information regarding the protocol used for
figure 5. A. Dark exposure of adult aphids: Aphids were raised in light/dark periods
(16/8 hours) then submitted to the following conditioning: adult aphids (1012 days
after birth for the green and 710 days for the orange aphids, according to their
temperature-dependent life cycle) were placed in dark, then the full population is
tested after two days (1 and 2; 5 and 6) for ATP content or alternatively were kept in
light two days more as control (3 and 4; 7 and 8) before the measure of ATP content.
ATP determination was also obtained with the content of one white aphid from a
cohort placed in the same conditions as the corresponding lane described above.
B. Dark exposure of larvae: Orange aphid mothers were raised in light/dark periods
(16/8 hours), then after birth the larvae were conditioned as indicated in legend to
figure. The standards roughly represent 50 pmoles (blue) to 250 pmoles (red).
C. Comparative time course of the decline of ATP content in embryos and larvae
placed in dark: The full ovarioles were dissected from orange aphid mothers kept in
dark according to the indicated timing, then analyzed for their ATP content. A
comparative determination was performed with the larvae placed in dark after birth
from orange aphid mothers raised in light. The determinations were conducted with
the ovarioles from three mothers and with five larvae.
D. Light-induced ATP synthesis after a dark exposure episode of orange adult
aphids:
1 to 8 are parallel experiments carried out with the descendants of a unique orange
founder mother. The determinations were normalized with protein content.
E. Comparative determination of ATP between green, orange and white adult
aphids in dark or light: The determinations were carried out with the ATP extraction
from ten adult aphids for each experiment. Determinations were normalized with
protein content.
F. Light-induced ATPsynthesis after a dark exposure episode in larvae: Top: orange
mothers were placed in dark and first instar larvae were tested at day 0 (1), at day 2 (2)
and day 3 (3). Down: adult orange mothers were placed three days in dark, then placed
back in light (day 0). Emerged progenies (first instar larva) were tested at day 0 (1), at
day 2 (2) and finally at day 3 (3). Determinations were normalized with protein
content. Standards roughly represent 20 pmoles (green) to 100 pmoles (red).
Figure 7 | NAD
1
/NADHbalance incytosol and particulate (mitochondria) fractions indark- and light-conditioned aphids. The dosage was performed
with orange (A) and white (B) aphids conditioned in light or dark using methyphenylsulfonate and tetrazolium salts (see Methods). Briefly, 50 adult
aphids (orange and white phenotypes), kept in dark for three days or alternatively kept in light photoperiodicity (18/6 hours), were grinded and assayed
for the NAD
1
/NADH balance. The experiments were repeated three times. Bars are the mean 1/2S.E. **P,0.005. The Raman imaging between 0 to
100 mm in depth was performed and shows a double layer structure of carotenoid compounds (C). A control carotene signature in Raman imaging
corresponding to the analysis in (C) is shown in (D).
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SCIENTIFIC REPORTS | 2 : 579 | DOI: 10.1038/srep00579 7
Extraction of pigments for spectral analysis. Aphids were extracted with Ringers
buffer
27
(20 adult orange and green variants) in a glass potter and centrifuged 20 min
at 13,400 x g. The supernatants were normalized with the weight of the pellet and
submitted to spectral absorbance from 290 to 700 nm. We observed at the top of the
water phase an orange intense layer. Another orange layer, found at the top of the
pellet, was collected with a glass pipette in 100 ml of water and re submitted to
centrifugation. The orange layers at the top of the water phase were added. The crude
isolation of these orange pigments was facilitated by their known rapid crystallisation.
This orange precipitate after crystallisation was solubilized in acetone/ethanol (25/75)
and analyzed by spectrometry of absorbance. The obtained spectral absorbance
properties confirmed to be in accordance with carotenoid molecules (peak of
absorbance at 425, 450 and 480 nm). On the other hand, the green pigment was found
for two thirds in the membrane (hydrophobic compounds extracted by ethanol or
ether) and for one third in the soluble fraction (bound to proteins as hydrophobic
compounds or esterified with carbohydrates). The molecular structure of this green
pigment is still unknown due to its complexity (probable polyphenolic compound
28
).
Raman imaging spectrometry. The Raman analysis was conducted with living
animals. The equipment was a spectrophotometer Labram HR800 Horiba Jobin-
Yvon. An argon ion laser beam was focused on the sample by using a 100x objective
(NA 0.9) for crystal analyses and a 50x LWD objective (NA50.45) for aphid analyses
and Raman back scattered light was collected by the same objectives. Then, we
estimated the analysed area to about 1 square mm with 100x and about 10 square mm
with 50x.
Extraction of carotenoids for spectrometry analysis. Carotenoid extraction was
adapted from previous work
29,30
. One gram of aphids previously milled in liquid N
2
was added to 80 mg of MgCO
3
in 15 ml of extraction solvent (ethanol/hexane, 4:3
v/v, containing 0.1% of BHT as antioxidant) and stirred for 5 min. The residue was
separated from the liquid phase by filtration with a filter funnel (porosity Nu. 2) and
washed successively with 15 ml of the above solvent, 15 mL of ethanol and 15 mL of
hexane. Organic phases were transferred to a separating funnel and successively
washed with 40 mL of 10% sodium chloride and 2 x 40 mL of distilled water. The
aqueous layer was removed. The hexanic phase was dried under anhydrous sodium
sulphate, filtered and evaporated to dryness at 40uC in a rotary evaporator. The
residue was dissolved in 250 mL dichloromethane and 250 mL MTBE/methanol
(80:20, v/v). Samples were placed in amber vials before chromatographic analysis.
Liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS)
analysis of carotenoids. The HPLCapparatus was a Surveyor plus model equipped of
an autosampler, a PDA detector and LC pumps (Thermo Electron Corporation, San
Jose, CA, USA). Carotenoids were analysed according to previously published
methods
31,32
. Carotenoids were separated along a C30 column (250 34.6 mm, 5 mm
particle size), YMC (EUROP, GmbH). The mobile phases were water/20 mM
ammoniumacetate as eluent A, methanol/20 mMammoniumacetate as eluent Band
MTBE as eluent C. Flowrate was fixed at 1 mL/min and the column temperature was
set at 25uC. A gradient program was performed: 02 min, 40% A/60% B, isocratic
elution; 25 min, 20% A/80% B; 510 min, 4% A/81% B/15% C; 1060 min, 4%
A/11% B/ 85% C; 6071 min, 100% B; 7172 min, back to the initial conditions for
re-equilibration. The injection volume was 10 mL and the detection was monitored
from250 to 600 nm. After passing through the flowcell of the diode array detector the
column eluate was split and 0.5 ml were directed to the ion trap of a LCQ mass
spectrometer fitted with an electrospray interface (Thermo Finnigan, San Jose,
California, USA). Experiments were performed in positive ion mode. Scan range was
1002000 and scan rate 1 scan/s. The desolvation temperature was set at 25uC.
High performance liquid chromatography analysis of carotenoids. Carotenoids
were analysed by HPLC using an Agilent 1100 System (Massy, France). The column
and gradient conditions were the same as used in mass spectrometry analysis. The
injection volume was 20 mL. Absorbance was followed at 290, 350, 400, 450 and
470 nm using an Agilent 1100 photodiode array detector. An Agilent Chemstation
Plus software was used for data analysis. Each analysis was made in triplicates. All
carotenoid concentrations were expressed in b-carotene equivalent.
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Acknowledgements
We thank Patrick Coquillard and Rene Feyereisen for support, advice and discussions that
have contributed to design experiments and George De Sousa for help with ATP dosage. We
are grateful to Angela Algeri for critical reading of the manuscript. The work was funded by
an ANR grant (ANR-06- Blan:NT05-2_43193) and a CNRS grant (PEPS 20092437). This
study was facilitated by the sequencing of the aphid A. pisum genome conducted by the
authorss Consortium and carried out at Baylor College of Medicine (Houston, TX, USA).
Author contributions statements
J. C. V. performed the Raman experiments, A. D. did the selection of aphid variants, P. B.
and C. M. did the HPLC and mass spectrum analysis of carotene, J. C. V., P. B. and A. R.
designed the experiments, M. C. contributed to the experimental work, J. C. V., M. C. and
A. R. wrote the manuscript.
Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
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Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this
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How to cite this article: Valmalette, J.C. et al. Light- induced electron transfer and ATP
synthesis in a carotene synthesizing insect. Sci. Rep. 2, 579; DOI:10.1038/srep00579 (2012).
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SCIENTIFIC REPORTS | 2 : 579 | DOI: 10.1038/srep00579 8